UV222 disinfection of SARS-CoV-2 in solution

There is an urgent need for evidence-based engineering controls to reduce transmission of SARS-CoV-2, which causes COVID-19. Although ultraviolet (UV) light is known to inactivate coronaviruses, conventional UV lamps contain toxic mercury and emit wavelengths (254 nm) that are more hazardous to humans than krypton chlorine excimer lamps emitting 222 nm (UV222). Here we used culture and molecular assays to provide the first dose response for SARS-CoV-2 solution exposed to UV222. Culture assays (plaque infectivity to Vero host) demonstrated more than 99.99% disinfection of SARS-CoV-2 after a UV222 dose of 8 mJ/cm2 (pseudo-first order rate constant = 0.64 cm2/mJ). Immediately after UV222 treatment, RT-qPCR assays targeting the nucleocapsid (N) gene demonstrated ~ 10% contribution of N gene damage to disinfection kinetics, and an ELISA assay targeting the N protein demonstrated no contribution of N protein damage to disinfection kinetics. Molecular results suggest other gene and protein damage contributed more to disinfection. After 3 days incubation with host cells, RT-qPCR and ELISA kinetics of UV222 treated SARS-CoV-2 were similar to culture kinetics, suggesting validity of using molecular assays to measure UV disinfection without culture. These data provide quantitative disinfection kinetics which can inform implementation of UV222 for preventing transmission of COVID-19.

www.nature.com/scientificreports/ to inform engineering design decisions which balance the risk to eyes and skin from UV exposure with the risk of infection from virus transmission.
Here we demonstrate the dose response kinetics of SARS-CoV-2 in liquid after exposure to primarily 222 nm UV light emitted by a krypton-chlorine (KrCl) excimer lamp (excilamp) filtered to reduce transmission of more harmful wavelengths > 240 nm. The lower wavelength emission (222 nm) is neither carcinogenic in human skin models or rodents 12 , nor causes acute corneal damage in rodents 13 . Additionally, the 222 nm wavelength emitted by KrCl excilamps is inherently more effective at disinfection 14 , nucleic acid damage 15 , and protein damage 16,17 than 254 nm emitted by low pressure mercury lamps due to greater absorbance of target biomolecules at lower wavelengths. Krypton and chlorine in KrCl excilamps are much less toxic than mercury, and KrCl excilamps have already been shown to be competitive in terms of electrical efficiency with mercury lamps that have many more years of product development and optimization 18 . To provide a safer quantification of dose responses without requiring viral proliferation that is required in standard culture-based assays, damage to the nucleocapsid protein and N gene were measured after UV222 treatment using commercial assays. Our results demonstrate that when an aqueous solution of pathogenic SARS-CoV-2 is exposed to UV 222 light emitted by a Kr-Cl excilamp, its infectivity and integrity is attenuated in a UV dose-dependent manner, as measured by culture and molecular assays. These first UV 222 disinfection dose responses demonstrate the feasibility of UV as an approach to inactivate SARS-CoV-2.
Methods SARS-CoV-2 culture. SARS-CoV-2, Isolate USA-WA1/2020, was obtained from Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Batch # 70034262) and stored and cultured in the Ohio State University Biosafety Level 3 laboratory (IBC Protocol # 2020R00000046). The viral stock used in this study was established by thawing the Batch, diluting it 1:10,000 into incomplete DMEM (Gibco Cat# 11995-065, supplemented with 4.5 g/L d-glucose, 110 mg/L sodium pyruvate), and adding it to T175 flasks of confluent Vero cells (ATCC clone E6) for a one hour incubation period (37 °C, 5% CO 2 ), after which the supernatant was removed and replaced with complete DMEM (cDMEM; DMEM as above plus 4% heat-inactivated fetal bovine serum). These T175 flasks were incubated for 3 days (37 °C, 5% CO 2 ) to propagate infectious virus. At the end of this period, visual inspection of the flasks under a light microscope demonstrated that the nearly all Vero cells were dead. The supernatants in each of the T175 flasks were presumed to contain infectious virus at this point, were carefully transferred and combined into a 50 mL conical, centrifuged at low speed to remove cell debris, aliquoted into microcentrifuge tubes, frozen and stored at − 80 °C. The live virus titer in frozen aliquots was determined to be ~ 10 7 plaque forming unit (PFU) per mL using a modified version of plaque assay developed by the Diamond laboratory 19 and described below.
UV dose calculations. The UV 222 light source (USHIO Care222 ® ) is a KrCl excilamp that is optically filtered to reduce emission > 240 nm. The UV source was turned on to warm up for 15 min before any irradiance or spectral measurements or irradiations. Standardized procedures were followed for carrying out quasi-collimated beam disinfection studies 20 and calculating polychromatic UV doses 21 . The emission spectrum of the UV 222 source was measured using a NIST-traceable calibrated Ocean Optics HDX UV-Vis spectroradiometer with an extreme solarization resistant 455 µ fiber and Spectralon diffusing cosine corrector detector. Raw spectral data from the OceanView software was interpolated to integer wavelengths using the FORECAST function in Microsoft Excel and relativized to peak emission at 222 nm for use in dose calculations ( Fig. 1 and Supplementary  Fig. S1). Total incident UV-C irradiance was measured using an International Light Technologies (ILT) 2400 radiometer with a SED 220/U solar blind detector, W Quartz wide eye diffuser for cosine correction, and peak irradiance response NIST-traceable calibration. For irradiance measurement, the peak wavelength calibration value was input manually as the radiometer factor. The incident irradiance was measured with the detection plane of the radiometer centered at the height and location of the sample surface during UV exposures, and corrected for several factors to determine the average irradiance through the sample depth. Spatial nonuniformity of emission was accounted for each test by measuring irradiance at 0.5 cm increments from the center to the edge of the petri dish and relativized to determine a petri factor, which was always > 0.9. The typical detector spectral response was obtained from ILT and used to calculate the radiometer factor integrated over the lamp emission, which was 0.9971. As previously 22 , the reflection factor for water at the 222 nm peak wavelength was assumed to be 0.9726. The divergence factor was determined each experiment day by accounting for the distance between the lamp and the sample surface, and the sample depth and was always > 0.9. The water factor was determined each sample day by the ratio between the incident irradiance and the average irradiance integrated through the sample depth after wavelength-specific absorption. The UV-vis absorbance of virus working stocks (prepared fresh for each test) was measured in the biosafety cabinet using a Nanodrop™ One C spectrophotometer via the microvolume pedestal for wavelengths 200-295 nm and the 1 cm quartz cuvette for wavelengths above 195 nm. Working stock absorbance spectra for each test are shown in Figs. 1 and S1. After these adjustments to incident irradiance in the center of the sample, the average irradiance was used to calculate exposure times (max: 15 min; min: 15 s) for pre-determined UV doses (0-40 mJ/cm 2 ). Three disinfection tests were performed with exposure times up to 115 s for UV doses up to 2.7 mJ/cm 2 , up to 856 s for UV doses up to 40 mJ/cm 2 , and up to 1260 s for UV doses up to 30 mJ/cm 2 , respectively. (Summarized in Supplementary Table S1).

UV treatment.
All UV measurements, sample preparation, UV treatments, and subsequent handling of treated samples were performed in a biosafety cabinet. On the day of each three biologically independent tests while the UV source warmed up and measurements were taken for dose calculations, aliquots of SARS-CoV-2 (previously tittered at 10 7 PFU/mL) were diluted in cDMEM to make a "working stock solution" with a target www.nature.com/scientificreports/ titer of 10 5 PFU/mL. For each UV dose tested, 3 mL of the working stock solution was pipetted into a 3.7 cm 2 area and 3.5 cm diameter polystyrene tissue culture dish (VWR Catalog # 82050-538) with a sterile Tefloncoated micro stir bar (VWR Catalog # 58948-353) and positioned under the UV light on a small stir plate to achieve quiescent mixing while blocking the UV light with a shutter. After removing the tissue culture dish lid, the shutter was removed to expose the sample to UV light for the calculated exposure time corresponding to the pre-determined UV dose before replacing the aperture to end the UV exposure. Immediately afterwards, the treated media was transferred to a sterile 15 mL polypropylene centrifuge tube (VWR) and used for the assays described below. Working stocks for untreated samples were placed on the stir plate for a representative amount of time with the lamp off before transfer to centrifuge tube (0 mJ/cm 2 ).
SARS-CoV-2 plaque assay. Plaque assays were used to determine PFU/mL of samples before UV treatment (0 mJ/cm 2 ) and after UV treatment (all other UV doses). The plaque assay used for this study is a modification of that which was originally developed and reported by Case et al. 19 and is listed here as STEPS 1-5. (STEP 1) At least 18 h prior to the assay, 12-well plates were seeded with a sufficient number of Vero cells so that each well was confluent by the assay start; plates were incubated overnight at 37 °C. www.nature.com/scientificreports/ the warmed cDMEM/methylcellulose mixture. (STEP 6) The culture plate was then returned to the incubator and left undisturbed for 3 days. On the final day (Day 3), cDMEM/methylcellulose mixture was removed from each well, cells were fixed with 4% para-formaldehyde in PBS (20 min, room temperature), washed with PBS and stained with 0.05% crystal violet (in 20% methanol). After rinsing plates with distilled water, plates were dried and plaques were counted under a light microscope at 20× magnification.

SARS-CoV-2 outgrowth assay.
The virus outgrowth assay used for this study is identical to the plaque assay described above, with the exception that after STEP 4 the virus laden media was replaced with 1 mL of warm cDMEM (instead of a cDMEM/methylcellulose mixture). Afterwards, the culture plate was returned to the incubator and left undisturbed for 3 days. On the final day, the cell supernatants of each well were collected, transferred into a microcentrifuge tube, centrifuged at low speed to remove cell debris (1000×g, 10 min), aliquoted into microcentrifuge tubes, frozen and stored at − 80 °C. Aliquots were subsequently used for quantitative real time PCR (qRT-PCR) measurement of SARS-CoV-2 nucleocapsid (N) gene copies, as well as ELISA determination of SARS-CoV-2 N protein concentrations.
SARS-CoV-2 N gene quantitation-N1 primer set. Quantitative PCR (qPCR) was used to quantify the SARS-CoV-2 N gene directly in RNA extracts of samples before UV treatment (0 mJ/cm 2 ) and after UV treatment (all other UV doses) ("Day 0" samples), and in RNA extracts of cell supernatant aliquots from outgrowth assays ("Day 3" samples). RNA was extracted from samples using the QIAamp Viral RNA method (Qiagen), and converted to cDNA using the SuperScript IV first strand synthesis method with random hexamer primers (Invitrogen). cDNA was subsequently amplified with the "N1 primer set" and associated PCR conditions that were originally developed by the Centers for Disease Control 23  . q-PCR assays were run on a BioRad CFX Connect Real Time PCR system to determine C T values from samples and standards. A standard curve was generated for the N1 primer set by running serial dilutions on each plate of in vitro transcribed RNA converted to cDNA relating N gene copy numbers to C T values. To generate this standard, RNA was extracted from an aliquot of our SARS-CoV-2 stock and converted to cDNA before amplification of the N gene using the N1 primer set as described above. The amplicon was visualized by agarose gel electrophoresis, gel extracted and cloned/ligated into the plasmid vector pCR II-TOPO (Invitrogen), downstream of the T7 promoter. Ligation products were transformed into E. coli, and mini-preps of randomly selected colonies were screened via PCR for the presence of insert. A single clone was then used to produce in vitro transcribed (IVT) N gene RNA-a reagent necessary for accurate gene copy number measurement-using the HiScribe T7 Quick High Yield RNA Synthesis method (New England Biolabs). After treating the IVT RNA with DNase and performing a cleanup reaction, the RNA concentration was determined via Nanodrop. The copies of single stranded N gene RNA transcripts per µL was determined by the following equation: [RNA concentration (Nanodrop measurement, ng/µL) × the Avogadro number (6.02 × 10 23 )]/[Predicted molecular weight of transcript (23 kDa) × 10 9 ]. Serial dilutions of IVT RNA were made (range: 10 13 ⟶10 -1 copies/µL), converted to cDNA as above and used as standards in the N gene copy number assay described above.  www.nature.com/scientificreports/ regression coefficients) were performed using these programs' bundled software. Log 10 Reduction (LR) was calculated as log 10 (N o /N), where N was viral PFU/mL in the plaque assay, N gene copies/µL in qPCR assays for either the short N1 amplicon or the long N1-2 amplicon, or N protein concentration in pg/mL in the ELISA assay after exposure to a given UV 222 dose, and N o was the initial concentration. The level of replication in this study was three biologically independent tests, with at least technical duplicates for each assay. The summary of biological and technical replicates for all the culture and molecular assays are shown in Table S2.

SARS-CoV-2 infectivity response to UV 222 . Viral infectivity UV 222 dose response was characterized
by exponential decay kinetics (Fig. 2). Representative plaque assay results for Experiment 2 are shown in Supplementary Fig. S2. At a mean initial viral titer of 6.51 × 10 4 PFU/mL, the pseudo first order rate constant for viral disinfection was − 1.48 cm 2 /mJ (R 2 = 0.89). When expressed as log 10 reduction (LR) of viral infectivity after exposure to a given UV dose, the linear rate constant was 0.64 cm 2 /mJ (R 2 = 0.95), which equates to a D 90 (dose for 1 log 10 or 90% inactivation) = 1.6 mJ/cm 2 . Doses ranges and initial Vero cell confluence were only sufficient in the Test 3 experimental replicate to quantify a dose response. However, in Test 2, the mean initial viral titer of 3.54 × 10 4 PFU/mL in untreated samples was reduced to below detection by the first dose tested of 10 mJ/cm 2 , equivalent to a LR of at least 4.25 logs. These results were also consistent with qualitative results from Test 1, www.nature.com/scientificreports/ where Vero cells appeared mostly dead in the untreated samples, appeared increasingly healthy through doses 0.7 and 1.4 mJ/cm 2 , and appeared healthy at doses above 2 mJ/cm 2 .

SARS-CoV-2 N gene and protein response to UV 222 . Across all tests, detection of SARS-CoV-2 in the
N1 assay was 10.66 ± 0.27 log 10 copies/µL in cell cultures infected with untreated virus (0 mJ/cm 2 ), 5.06 ± 0.78 log 10 copies/µL in uninfected cell culture supernatants, 5.49 log 10 copies/µL in RNA extraction negative control, 3.66 ± 0.23 log 10 copies/µL in no template RT-qPCR reaction controls (concentration data and standard curves shown in Supplementary Figs. S3 and S5). Due to amplification in RNA extraction negative controls (CT values listed in Table S3), we excluded points with CT values greater than CT values of the RNA extraction negative control for samples, control samples, and standard curves. Despite this background, dose responses were still discernable because background is canceled out by the LR calculation. Additionally, Day 3 outgrowth assay data demonstrated a nearly 3 log 10 copies/µL signal increase in cell cultures infected with untreated virus (0 mJ/cm 2 ), ranging from 9.7 to 9.9 log 10 copies/µL on Day 0 to 12-12.6 log 10 copies/µL on Day 3. This increases the ability to detect lower initial concentrations due to propagation of infectious virus, and consequently decreases the LR detection limit to provide a better estimation of the UV dose response. For the short amplicon spanning the N1 region of the N gene (CDC 2019), viral RNA damage in response to UV 222 immediately after treatment ("Day 0") was also characterized by exponential decay kinetics (Fig. 3A). When expressed as LR of N1 copies/µL in qPCR reactions after exposure to a given UV dose, the linear rate constant was 0.049 ± 0.005 cm 2 /mJ (slope ± standard error, R 2 = 0.92). The N1 dose response was modeled using the linear region between 0 and 20 mJ/cm 2 to avoid tailing in the dose response. When including only doses up to 10 mJ/cm 2 as for the plaque assay, the slope (0.07) of the N1 gene damage dose response at Day 0 was higher than doses up to 20 mJ/cm 2 (0.05) while R 2 was the same (0.92). The dose response curve between 0-10 mJ/ cm 2 and between 0-20 mJ/cm 2 are shown in Figs. S4 and 3A separately. Compared with the LR rate constant of SARS-CoV-2 infectivity measured by plaque assay, the LR rate constant of N gene damage measured by N1 qPCR was approximately tenfold lower.
For the N1 dose response after 3 days in the outgrowth assay ("Day 3") for doses up to 0-20 mJ/cm 2 in Fig. 3A, the linear rate constant was 0.230 ± 0.033 cm 2 /mJ (slope ± standard error, R 2 = 0.78). Although a positive dose response was apparent and the slope was closer to the plaque assay (indicating better ability to predict plaque assay dose response with combined cell culture with qPCR), the increased variability introduced by cell culture decreased the strength of the regression.
For the long amplicon spanning both the N1 and N2 regions of N gene (CDC 2019), viral RNA damage in response to UV 222 immediately after treatment ("Day 0") was also characterized by exponential decay (Fig. 3B). The linear rate constant for LR versus UV 222 dose was 0.056 ± 0.005 (slope ± standard error, R 2 = 0.94). Compared with the LR rate constant for of SARS-CoV-2 infectivity measured by plaque assay, the LR rate constant of N gene damage measured by N1-2 qPCR was approximately tenfold lower. This similarity indicates that increasing the amplicon length did not increase the ability to detect gene damage that correlates with loss of viral infectivity. Across all tests, the positive signal for SARS-CoV-2 in the N1-2 assay was 4.7 ± 0.1 log 10 copies/µL in cell cultures infected with untreated virus, undetected in uninfected cell culture supernatants, and 0.08 ± 1.4 copies/ µL in no template RT-qPCR reaction controls (concentration data and standard curves shown in Supplementary  Figs. S3 and S5). Because the long amplicon assay was used to investigate potential for improved measurement of disinfection dose response without culture, no Day 3 samples were analyzed.
For Fig. 3C, although no dose response was observed for LR of the N protein versus UV 222 dose immediately after treatment ("Day 0") for doses up to 40 mJ/cm 2 (0.002 ± 0.001 cm 2 /mJ, slope ± standard error, R 2 = 0.21), a stronger dose response was observed in Day 3 cell culture supernatants for doses up to 20 mJ/cm 2 (0.243 ± 0.028 cm 2 /mJ, slope ± standard error, R 2 = 0.21) (Fig. 3C). Across all tests, the positive signal for SARS-CoV-2 in the N protein assay was 2.69 × 10 5 ± 9.83 × 10 4 pg/mL in untreated virus samples on Day 0, 1.41 × 10 8 ± 2.99 × 10 8 in Day 3 cell culture supernatants infected with untreated virus, and below detection in uninfected cell culture supernatants (concentration data and standard curves shown in Supplementary Figs. S3 and S5).

Discussion
This study provides the first rigorous UV 222 dose response kinetics for SARS-CoV-2 in aqueous solution, but there are limitations that must be acknowledged. Most importantly, this study was conducted using virions suspended in aqueous solution. This is only a starting point for quantifying dose response kinetics for airborne virus disinfection that is most relevant for this virus, where many factors such as temperature, humidity, air flow dynamics, and UV reactor specifics will impact dose responses. Previous studies comparing disinfection kinetics of infectious agents in air at increasing relative humidity to those in water [24][25][26][27][28][29] indicate that these water dose responses may present a conservative estimate of airborne disinfection kinetics because humidity in many indoor environments is conditioned to reduce infectious agent persistence.
One additional limitation of this study related to UV 222 application in indoor environments is that the disinfection impact of any ozone production by vacuum UV wavelengths potentially emitted by the KrCl excilamp was not measured, but can likely be neglected due to high airflows in the biosafety cabinet and BSL3 facility. The negative air quality impacts and building material degradation by ozone potentially generated by these lamps, and the potential health hazards and building material solarization from wavelengths below 240 nm and the nonzero emission at wavelengths above 240 nm ( Supplementary Fig. S1), should also be considered when weighing the benefits of reducing infectious disease transmission by UV 222 for COVID-19 and other infectious diseases.
Considering these limitations, these data provide a strong foundation for future development and application of UV 222 for reducing airborne viral transmission. UV 222 is at least 4.2 times safer for human exposure (the threshold limit values for human UV exposure before recent updates were 25 mJ/cm 2 and 6 mJ/cm 2 at 222 and    34 . The linear constant rate of 1.42 cm 2 /mJ is higher than the value reported by our study. This could be explained by our high sample absorbance at 222 nm compared to theirs (0.05 cm −1 ), their different experimental setup testing disinfection in thin films rather than aqueous solution, or our dose response modeling that included the lowest dose to exceed the plaque assay detection limit (Fig. 2). A recent review 35 predicted the median D 90 for coronavirus disinfection by UV 254 to be 3.7 mJ/cm 2 . Our results and these predictions are in general agreement with recent UV 222 and UV 254 disinfection studies of SARS-CoV-2 as recently reviewed 29 . However, some of these studies are still in the process of peer review and/or did not use standardized UV disinfection procedures that allow comparisons between experiments and precise quantification of doses. In the only UV 222 SARS-CoV-2 surface decontamination study to date 36 , researchers report 0.94 LR after 10 s exposure to 0.1 mW/cm 2 . Although UV dose cannot be calculated for this study in the absence of sample absorbance and differences in experimental setup, these results demonstrate a high degree of susceptibility of SARS-CoV-2 to UV 222 and generally align with ours. Considering our data in context of literature, UV 222 is a promising disinfection method for SARS-CoV-2 in aqueous solution. These infectivity and molecular dose response data could immediately inform measures to prevent potential transmission by water or wastewater where infectious SARS-CoV-2 and other viruses have been shown to be potentially persistent for days [37][38][39] . Although tailing was observed in dose responses for molecular assays and may have been contributed from clumping of virus in the protein-laden growth media, viruses were disinfected below detection in plaque assays, indicating that aggregation did not interfere with complete viral inactivation. We did not observe a strong relationship between the kinetics of N gene damage (measured by qPCR with a short and long amplicon) and disinfection, which could reflect that protein damage contributes more to disinfection than genome damage for SARS-CoV-2. One study of MS2 bacteriophage found RNA genome damage to be closely related to and thus contributed to disinfection kinetics 15 , whereas a study of Adenovirus found DNA genome damage not to be closely related to disinfection 40 . This disparity between these viruses with different structures and hosts was further demonstrated when it was shown that protein damage, especially to external capsid proteins, contributes more strongly to UV disinfection of Adenovirus 41 . However, we also did not see a strong association between the kinetics of N protein damage and disinfection. Because we only measured the N protein that closely associates with the viral genome, we may have missed damage to external proteins such as the spike protein which are on the surface to absorb incoming UV radiation and are vital in infection of host cells 42 . Understanding impacts of UV 222 on the spike protein was not possible when conducting this study due to limited assay availability, but will continue to be important as SARS-CoV-2 variants with spike protein mutations continue to emerge. Additionally, the confirmation and sequence of the genome and proteins can affect UV genetic damage [43][44][45][46][47] , so the N protein and gene may not be the targets that primarily contribute to disinfection-inducing molecular damage. These factors could explain the weak relationships we observed between disinfection kinetics and N gene damage or N protein damage, and warrant further investigation to unravel the mechanisms of disinfection at this and other UV wavelengths. While these mechanistic complexities remain to be resolved, the disinfection kinetics we report indicate the high degree of susceptibility of SARS-CoV-2 in aqueous solution to UV 222 .

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.